Ghrelin is a peptide which was isolated from the stomach but is expressed also in many other tissues, including the endocrine pancreas. It was discovered as a natural ligand of the growth-hormone secretagogue receptor type 1a (GHS-R) (Refs. 1, 2). Ghrelin acylation at serine 3 is essential for binding to GHS-R1a, which mediates GH-releasing activity and also the orexigenic action of acylated ghrelin. Besides stimulating GH secretion and modulating other pituitary functions, acylated ghrelin (AG) exerts a broad range of biological actions such as central regulation of food intake and energy balance and control of insulin secretion and glucose metabolism. GHS-R1a expression has been detected in a variety of endocrine and non-endocrine, central and peripheral animal and human tissues, including the pancreas. Notably, the link between ghrelin and insulin seems of major relevance. AG has been shown to possess hyperglycemic diabetogenic effects; ghrelin knock-out mice display enhanced glucose-induced insulin release while blockade of pancreatic islet-derived ghrelin has been shown to enhance insulin secretion and to prevent high-fat diet-induced glucose intolerance in rats.
In the endocrine pancreas, ghrelin has been shown to localize to α- and β-cells and to the newly identified ghrelin-producing islet ε-cells, suggesting a role in the regulation of β-cell fate and function (Refs. 9, 22, 19). Survival of β-cells is of major importance for maintaining normal glucose metabolism and β-cell apoptosis is a critical event in both type 1 and 2 diabetes (Refs. 16, 21).
Studies with several analogs of ghrelin with various aliphatic or aromatic groups in the side chain of residues 3 and several short peptides derived from ghrelin with various aliphatic or aromatic groups in the side chain of residue 3 and several short peptides derived from ghrelin as well showed that bulky hydrophobic groups in the side chain are essential for maximum agonist activity. In addition, short peptides encompassing the first four or five residues of ghrelin were found to functionally activate GHS-R 1a about as efficiently as the full-length ghrelin. Thus, the entire sequence of ghrelin is not necessary for activity; the Gly-Ser-Ser(n-octanoyl)-Phe [SEQ ID NO: 29] segment appears to constitute the active core required for agonist potency at GHS-R 1a.” (Ref 2)
Unacylated ghrelin (UAG) is the major circulating form of ghrelin and has long been believed to be biologically inactive since it does not bind GHS-R1a at physiological concentrations and is thus devoid of GH-releasing activity. It is now known that UAG is a biologically active peptide, particularly at the metabolic level, having notably been shown to exert anti-diabetogenic effects as described in U.S. patent application Ser. No. 10/499,376, published on Apr. 14, 2005, under publication number US 2005-0080007. Indeed UAG is able to: a) counteract the hyperglycemic effect of AG in humans (Ref. 6); b) directly modulate glucose metabolism at the hepatic level by blocking basal, glucagon-induced and acylated ghrelin-stimulated glucose output from hepatocytes (Ref. 3); c) decrease fat deposition, food consumption, and glucose levels in UAG transgenic animals (Ref. 7); d) stimulate proliferation and prevent cell death and apoptosis in β-cells and human pancreatic islets (Ref. 4).
It has recently been demonstrated that UAG is able to stimulate proliferation and to prevent cell death and apoptosis induced by (IFN)-γ/tumor necrosis (TNF)-α, synergism in β-cells and human pancreatic islets (Ref. 4). Noteworthy, cytokine synergism is considered to be a major cause for β-cell destruction in type I diabetes as well as of β-cell loss in type 2 diabetes. Moreover, this work also showed that UAG stimulated glucose-induced insulin secretion from β-cells that do not express GHS-R1a.
Together, these results reinforce the concept that UAG has a therapeutic potential in medical conditions associated with metabolic disorder such as conditions characterized by insulin deficiencies or by insulin resistance, including, but not limited to diabetes, and the effect of UAG on the β-cells is one of the mechanisms of action of UAG in these potential applications.
Recently, the therapeutic potential of UAG was clinically demonstrated, as a continuous infusion of UAG in healthy volunteers resulted in a lowering of blood glucose, an improvement in insulin sensitivity, a reduction in blood free fatty acids, and decreased cortisol levels.
UAG is a 28 amino-acid peptide and would preferably be administered to patients by intravenous or subcutaneous injection in order to produce its effects, which is not a convenient way to administer a drug to a patient. Also, peptides of this size are usually rapidly degraded following administration and their in vivo efficacy is often weak following intravenous, subcutaneous or intramuscular bolus administration.
In addition, manufacturing a 28 amino-acid peptide is a long and expensive process, whether it is manufactured by solid-phase peptide synthesis or by recombinant technology. Finally, chronically treating patients with a long peptide such as UAG might represent safety risks for the patients in the form of immunogenicity. Raising neutralizing antibodies against a natural peptide is a potential major health risk for the patients.
Therefore, it would be highly desirable to identify smaller size peptides that would possess a comparable biological activity to UAG, but would be easier and less costly to manufacture.
It would be even more desirable that these smaller size peptides would have increased biological potency when compared with UAG.
Another advantage of these smaller size peptides would be that they would bear fewer immunogenicity risks for patients upon chronic and repeated administrations, and hence exhibit a better safety profile. They may have a better bioavailability than UAG, whatever the route of administration, and be suitable for more convenient routes of administration, such as, but not limited to, transdermal, pulmonary, intranasal or oral delivery, or may constitute a starting material for the design of peptide analogs or peptidomimetic molecules with a better oral bioavailability. Smaller size peptides may also be compatible with drug delivery system such as, but not limited to, polymer-based depot formulations.